Determine if Epigallocatechin-3-Gallate has antibacterial effects on Acinetobacter baumannii and if so, determine its mechanism of action.

D.1. General Overview and Rationale

Acinetobacter baumannii is gram-negative bacteria that commonly isolated from hospital environment and hospitalized patients. This bacterium is capable of causing infection in organ transports and febrile neutropenia as well as associated with variety of diseases such as bacteremia, pneumonia, meningitis, wound, and urinary trace infection. Treating patients infected with A.baumannii has become a very difficult and challenging for clinics due to it is ability to withstand the antimicrobial power and that leads to develop new antibiotic resistant. However, the Epigallocatachin-3-Gallate …show more content…
Approaches and Methods

Bacterial Strains and Growth Conditions

The ATCC 19606 type culture was obtained from ________ and the five clinical isolates were obtained from____________. These clinical isolates are multi-drug resistant strains. All bacterial strains were grown on Luria-Bertani agar at 37°C. Also, each experiment requires the bacterial strain to grow overnight in Luria-Bertani broth. In addition, all antibiotics and Epigallocatechin-3-Gallate compounds were purchased from Sigma-Aldrich (St. Louis, MO) and appropriate concentrations were augmented in the culture media when needed.

Antimicrobial Susceptibility Testing of EGCG

The antibiotic susceptibility test is used to determine the minimal inhibitory concentration (MICs) using the broth dilution method. Serial two-fold dilutions of EGCG were performed in Mueller-Hinton Broth (pH 7.0). Also, the Minimal bactericidal concentrations (MBCs) were used to determine the lowest concentration of antibacterial agent that reduces the viability of the initial bacterial inoculum by ≥99.9% …show more content…
It was performed in duration of 12 hours. At 0 hours of the assay, cells were inoculated in four different conditions. The first condition is the control which wan only Mueller-Hinton Broth (pH 7.0). The second condition consisted of Mueller-Hinton Broth (pH 7.0) and 16 μg/mL of carbenicillin. The third condition was Mueller-Hinton Broth (pH 7.0) and 128 μg/mL of EGCG. The fourth condition consisted of Mueller-Hinton Broth (pH 7.0), 16 μg/mL of carbenicillin, and 128 μg/mL of EGCG. These solutions were incubated at 37°C. Every 4 hours, 100 μl samples from each condition were plated on Luria-Bertani agar. After a 16 to 18 hour incubation period at 37°C, the number of colonies forming units were measured manually. Time kill assay was also performed with another antibiotic which is meropenem to determine bacterial activity of A. baumannii clinical isolates. Sam procedure was used for meropenem, however, a concentration of 4 μg/mL of meropenem was used to determine bactericidal activity of A. baumannii clinical